Single base substitutions in human genes are the cause or are strongly associated with a variety of human diseases such as the hemoglobinopathies (Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim [1985] Science 230: 1350-1354; Embury, S. H., S. J. Scharf, R. K. Saiki, M. A. Gholson, M. Colbus, N. Arnheim, and H. A. Erlich [1987] N. Engl. J. Med. 316: 656-660) and cancer (Liu, E., B. Hjelle, R. Morgan, F. Hecht, and J. M. Bishop [1987] Nature 330: 186-188; Rodenhuis, S., M. L. van de Werering, W. J. Mooi, S. G. Evers, N. van Zandwijk, and J. L. Bos [1987] N. Engl. J. Med. 317: 929-935). Previously, if no convenient restriction sites were altered by the base change, then the only recourse has been to clone and sequence the affected gene. Recently, polymerase chain reaction (PCR) amplification of the DNA segment in question, coupled with hybridization of specific oligonucleotide probes, has allowed sequence determination without the need for molecular cloning. The applicability of the latter technique is dependent on the availability of versatile and inexpensive oligonucleotide probes.
Methods to covalently attach labels and reporter molecules to oligonucleotides have permitted their use as non-radioactive hybridization probes. New technologies in non-isotopic gene probes (Agrawal, S., C. Christodoulou, and M. J. Gait [1986] Nucl. Acids Res. 14: 6227-6245; Connolly, B. A. [1987] Nucl. Acids Res. 15: 3131-3139; Jablonski, E., E. W. Moomaw, R. H. Tullis, and J. L. Ruth [1986] Nucl. Acids Res. 14: 6115-6128; Haralambidis, J., M. Chai, and G. W. Tregear [1987] Nucl. Acids Res. 15: 4857-4876; Li, P., P. P. Medon, D. C. Skingle, J. A. Lanser, and R. H. Symons [1987] Nucl. Acids Res. 15: 5275-5287), DNA sequencing analysis (Smith, L. M., S. Fung, M. W. Hunkapiller, T. J. Hunkapiller, and L. E. Hood [1985] Nucl. Acids Res. 13: 2399-2412; Sproat, B. S., B. Beijer, P. Rider, and P. Neuner [1987] Nucl. Acids Res. 15: 4837-4848; Ansorge, W., B. Sproat, J. Stegemann, C. Schwager, and M. Zenke [1987] Nucl. Acids Res. 15: 4593-4602), electron microscopy (Sproat, B. S., B. Beijer, and P. Rider [1987] Nucl. Acids Res. 15: 6181-6196), and X-ray crystallography (Sproat et al. [1987] Nucl. Acids Res. 15: 4837- 4848) have provided impetus for the development and improvement of such methods. As applications continue to emerge, more convenient oligonucleotide labeling techniques and reagents will be required.
Current methods to introduce chemical modifications into oligonucleotides employ special phosphoramidite reagents during solid phase synthesis. Attention has focused on the 5' terminus and a number of protected amino-alkyl phosphoramidites have been reported (Agrawal et al., supra; Connolly, supra; Jablonski et al., supra; Smith et al., supra; Sproat et al. [1987] Nucl. Acids Res. 15: 6181-6196; Sinha, N. D. and R. M. Cook [1988] Nucl. Acids Res. 16: 2659-2669) to incorporate a 5' terminal aliphatic primary amine. Oligonucleotides modified by these reagents can be subsequently derivatized with fluorophores, biotin, and other molecules. Similarly, phosphoramidite reagents have also been described which incorporate a thiol functionality on the 5' terminus (Sproat et al. [1987] Nucl. Acids Res. 15: 4837-4848; Ansorge et al., supra; Connolly, B. A. [1985] Nucl. Acids Res. 13: 4484-4502).
Techniques modifying the 3' terminus are inconvenient and tedious. Lemaitre et al. (Lemaitre, M., B. Bayard, and B. Lebleu [1987] Proc. Natl. Acad. Sci. USA 84: 648-652; Lemaitre, M., C. Bisbal, B. Bayard, and B. Lebleu [1987] Nucleosides and Nucleotides 6: 311-315) have described the attachment of a ribonucleoside to the 3' terminus of an oligonucleotide using T4 RNA ligase. Terminal 3' modification was achieved after periodate-oxidation of the ribose ring followed by reductive amination. Another procedure by Zuckerman et al. (Zuckerman, R., D. Corey, and P. Schultz [1987] Nucl. Acids Res. 15: 5305-5321) incorporates a 3' terminal thiol group via solid phase oligonucleotide synthesis. Although this procedure is more efficient, it requires many synthetic steps and purifications. Thus, there remains a need for a simple and efficient method to synthesize 3' labeled oligonucleotides.